Marine microalgae co-cultured with floc-forming bacterium: Insight into growth and lipid productivity

Isolation and identification of microalgae

Biofloc samples were collected from water in ponds used to culture Whiteleg shrimp, L. vannamei. Centrifugation and agar plate streaking techniques were used to obtain a pure culture of the microalgae species used in this study (Phang & Chu, 1999; Okomoda et al., 2021). In brief, the samples were washed four times by centrifugation at 9,000×g rpm for 15 min. After each wash, the supernatant was discarded and the residue (i.e. pellet) resuspended, filtered through CF/C filter paper, and resuspended using distilled water. This process was performed to separate the microalgal cells from undesirable particles and microorganisms (McCurdy & Hodgson, 1974; Badar, Yaakob & Timmiati, 2017; Berthold et al., 2019). Thereafter, Conway medium agar (Tompkins et al., 1995) was prepared and poured into sterilized Petri dishes (100 × 15 mm diameter). Using a sterilized pipette, the microalgae residue suspension was dropped into each Petri dish, then spread over the agar using a sterile platinum hook. The Petri dishes were placed in an upside-down position, sealed with Parafilm tape, and incubated for seven days under laboratory conditions. Samples of the colonies that formed were transferred to slides and observed under a compound microscope. Selection of the different colonies was according to their size and colour. Observed mixed cultures were sub-cultured (streaked) to obtain pure cultures of the different microalgal species.

Colonies were observed under a microscope to ensure there was no cross-contamination of the microalgal species. Isolates were identified following the method described by Hasle et al. (1997). The axenic microalgae colonies were then incubated in 20 mL of saltwater enriched with Conway medium; thereafter, they were sub-cultured in 50 and 100 mL medium. Incubation conditions were maintained at 24 °C, 30 ppt salinity, 12:12 light-dark photoperiod (1,200 Lux), and 24 h aeration. In addition, every two days the cultures were examined using a microscope to ensure there was no cross-contamination of microalgal species or bacteria contamination in the inoculum. When the exponential growth phase was attained, inoculation of the microalgae was performed for both the axenic and co-culture treatments.

Experimental set-up and performance evaluation

Microalgae inoculum was produced for two treatments, namely; axenic single strain microalgae (i.e. control treatment) and axenic microalgae co-cultured with B. infantis. The B. infantis was obtained from the bioflocculation-bacteria collection at the Institute of Tropical Aquaculture and Fisheries (AKUATROP), University Malaysia Terengganu. To establish the axenic control treatment 50 mL of microalgae inoculum was mixed with 450 mL of sterilized saltwater in a 1 L Erlenmeyer flask. For the co-culture treatment, however, the inoculum ratio of microalgae to bacteria was determined at 9:1 (v/v); so the 1 L Erlenmeyer flask contained 450 mL of the sterilized saltwater, 45 mL of the microalgae inoculum, and five mL of the bacteria. The initial cell number of microalgae used ranged between 1.18 × 10⁶–2.0 × 10⁶ cell/mL. All the treatment groups were prepared in five replicates and incubated under the same environmental condition as described in “Isolation and Identification of Microalgae” (i.e. temperature = 24 °C; salinity = 30 ppt; photoperiod = 12:12 light-dark; photoperiod intensity = 1,200 Lux; and 24 h aeration).

To measure microalgae growth, the specific growth rate of microalgae was calculated based on concentration of chlorophyll a (Chl-a). A total of 10 mL samples were collected from the pure culture and co-culture experiment flasks and filtered through GF/C filter papers, followed by extraction in five mL acetone. Chl-a was thereafter quantified using a wavelength of 630, 645 and 665 nm in a UV-vis spectrophotometer (Strickland & Parsons, 1968). Biomass productivity, P (mg/L day), was calculated from the variation in Chl-a (mg/L) within the cultivation time. Specific growth rate (µ day⁻¹) was calculated using the equation given by Latiffi et al. (2017), as shown below:

$$\mathrm{S}\mathrm{p}\mathrm{e}\mathrm{c}\mathrm{i}\mathrm{f}\mathrm{i}\mathrm{c}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{w}\mathrm{t}\mathrm{h}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}(\mu \mathrm{d}\mathrm{a}{\mathrm{y}}^{-1})=ln\left(X_1/X_0\right)/\left(t_1-t_0\right)$$

Where X₁ and X₀ represent the growth of algae (mg/L) at days t₁ and t₀ respectively.

To determine the biomass concentration in terms of dried weight, a 50 mL sample of microalgae in the growth medium from each replicate was washed twice with distilled water and filtered each time through CF/C filter paper (Allen, 1952; Droop, 1969; McCurdy & Hodgson, 1974). Thereafter it was dried in an oven at 105 °C for 24 h to constant weight This method has proven effective at separating microalgae cells from bacteria in co-culture experiments (Badar, Yaakob & Timmiati, 2017; Contreras-Angulo et al., 2019; Berthold et al., 2019; Zhou et al., 2020).

Lipid was extracted through a modification of the method of Bligh & Dyer (1959), according to the report of Phang et al. (2015). In brief, soon after determination of the biomass concentration (Badar, Yaakob & Timmiati, 2017), the sample was ground and homogenized in 2:1 methanol: chloroform solution. The mixture was then centrifuged, and the supernatant was collected. The process was repeated twice to extract any remaining fat from the mixture. Thereafter, the supernatant was mixed with two mL of chloroform and washed with two mL of distilled water by vortexing and centrifugation for 10 min. The lower layer of the mixture was collected and blow-dried under nitrogen gas. After drying, the sample was re-dissolved in one mL chloroform and blow-dried again. Blow-drying was repeated before the sample was cooled in a desiccator containing silica gel for 24 h. Lipid content (%) of each sample, based on dried cell weight, was estimated using an electronic balance. Microalgae lipid productivity (in mg/L day) was calculated only at the exponential stage of growth, by dividing the obtained lipid content by volume of culture and by the duration of cultivation. The choice of time for the evaluation of the lipid content was based on the results of an earlier optimization.

The density of B. infantis was also quantified during the experiment, following the method described by Han et al. (2016). In brief, one mL of the culture was collected from the experimental flask daily and diluted tenfold. One tenth (i.e. one mL) of the dilution was then mixed with 25 mL of TSA solid medium and cultured at 30 °C for 48 h. Plates with 30–300 colonies were then selected in order to calculate the bacterial density, based on colony-forming units (CFU).

Statistical analysis

Descriptive statistics for Chl-a, specific growth rate, biomass concentration (DW), and lipid content were obtained using the software Minitab version 18.0. Statistical analysis of the parameters Chl-a, specific growth rate, and biomass concentration (DW) between treatments and days of cultivation were determined using two-way ANOVA and Tukey test at a significance level of 0.05 (Tybout et al., 2001). The mean differences of lipid content between the two treatments were analysed using the two sample t-test.

Microalgae isolates used

Six microalgae strains were identified in the biofloc sample; they included Tetraselmis sp., Nitzschia sp., Oscillatoria sp., Thalassiosira weissflogii, Chlamydomonas sp. and Chlorella vulgaris (Fig. 1). As shown in Fig. 2, low Chl-a concentrations and a rapid decrease in cell density observed in Tetraselmis sp., Nitzschia sp. and Oscillatoria sp. resulted in decreased cell growth during the acclimatization phase. However, the growth performance of T. weissflogii, Chlamydomonas sp. and C. vulgaris was better in the medium supplemented with Conway enrichment, and optimum Chl-a concentration was maintained during the inoculum production phase. As a result, only these three microalgae were used for the axenic/co-culture experiment in this study.

Growth of microalgae isolates in axenic condition and in co-culture

The growth profile of microalgae species in the axenic culture and in co-culture with B. infantis is presented in Fig. 3. Although the performance of T. weissflogii and Chlamydomonas sp. was similar in both treatments during the first two days, later a slight increase in Chl-a concentration was observed. For the C. vulgaris in the axenic condition, Chl-a concentration initially decreased (day 1), then recovered to a level above the initial concentration, and continued to increase until the sixth day. However, in co-culture conditions, Chl-a concentration of C. vulgaris was slightly reduced on day 3 and then increased afterward. In general, significantly higher growth was observed in the co-cultured microalgae species compared with those in axenic conditions (p ≤ 0.05).

The specific growth rate, as estimated between days 2 and 6 (for T. weissflogii) and between days 3 and 7 (for Chlamydomonas sp. and C. vulgaris), indicates the transition from an exponential growth stage to a stationary stage (Table 1). There were significant differences (p ≤ 0.05) in the specific growth rate of T. weissflogii between axenic and co-culture conditions; this indicates the occurrence of a rapid growth phase and achievement of peak (maximum) growth on days 4 and 2, respectively. T. weissflogii in axenic culture recorded a steady increase in biomass productivity up to day 6 (except day 5), however maximum biomass productivity of the co-culture treatment was achieved on day 4 (p ≤ 0.05). The specific growth rate of Chlamydomonas sp. in axenic and co-culture conditions achieved the maximal value on the sixth day. However, higher biomass productivity was observed in co-culture conditions compared to axenic conditions. C. vulgaris in co-culture conditions also attained maximum specific growth rate and maximum biomass productivity on the fourth day, before it started to decline.

The concentration of microalgae biomass is given by dry weight in Fig. 4. The biomass concentrations of microalgae obtained in the co-culture treatment was 20.32% higher than the biomass concentration obtained in axenic microalgae (p ≤ 0.05). These data indicate that the selected microalgae grew better in co-culture with B. infantis than in axenic conditions. Furthermore, T. weissflogii grown in co-culture showed a noticeable increase from day 4 to day 8 (p ≤ 0.05), compared to the axenic condition (Fig. 4A). Also, the maximum biomass concentration of Chlamydomonas sp. was observed on the fifth and seventh day for the axenic condition and co-culture condition, respectively (Fig. 4B). The biomass concentration of C. vulgaris, in contrast, increased steadily until the eighth day in both the co-culture and axenic treatments (Fig. 4C).

Lipid content of microalgae isolates in axenic condition and in co-culture

Microalgae lipid content was evaluated when the microalgae experienced the highest specific growth rate; hence, the lipid content of the microalgae at growth climax was evaluated as well as that after the climax point, as presented in Table 2. A decreasing trend of lipid content, post-growth climax, was observed in axenic conditions, while the converse was observed for the co-culture treatment (except for C. vulgaris). A similar trend was observed in the lipid productivity of the microalgae in this study. However, the biomass productivity of Chlamydomonas sp. was not significantly different among treatment and cultivation days.

Bacterial density in co-cultured microalgae species

The bacterial density of B. infantis in the co-cultured treatment is given in Fig. 5. The result showed significant variation in the cell density over time across the different microalgae tested. Peak CFU for B. infantis was observed in the co-cultured treatment with Chlamydomonas sp. and C. vulgaris on the third day of culture. Co-cultured T. weissflogii, on the other hand, consistently had a low number of CFU up until the fifth day when it started recording the highest values of all the treatments, until the eighth day (except for day 7, where performance was at the median level).